The transparent cornea is made up of an anterior stratified epithelium, a collagenous stromal matrix containing fibroblasts called keratocytes, Descemet's membrane and a single-layered endothelium. The major water-soluble proteins of the epithelium contain relatively few metabolic enzymes. In the mouse aldehyde dehydrogenase class 3 (ALDH3) and transketolase (TKT) comprise 30-50% of the water-soluble protein. This high concentration of metabolic enzymes is reminiscent of the "enzyme-crystallins" of the lens and suggests that they have a structural as well as catalytic role in the cornea. In the last few years we have cloned the mouse ALDH3 and TKT cDNAs and genes. Both are single-copy genes. The TKT gene is expressed at least 50 times more highly in the cornea than in other tissues, although it is essentially ubiquitously expressed throughout the body. The TKT gene is 30-40 kbp and utilizes two transcription initiation regions. The minor distal site is preceded by a TATA sequence, is utilized in the liver and is followed by an untranslated exon; the major transcription initiation region lies within intron 1, is used in the cornea and liver, lacks a TATA box, is GC-rich and initiates at multiple sites within a 10 bp span. The TKT gene contains a minimal -49/+90 promoter fragment when tested in cornea and lens cell lines by transfection utilizing the chloramphenicol acetyltransferase (CAT)reporter gene; higher promoter activity was obtained for the -510/+91 fragment. Transgenic mice using these transgenes showed very low expression in various tissues with no preferential expression in the cornea. The mouse ALDH3 gene is expressed at least 500 times higher in the cornea than other tissues, with low level expression detected in the stomach, urinary bladder, ocular lens and lung. Unlike TKT, corneal expression of ALDH3 is localized to the anterior epithelial cells and does not occur to any significant extent in the limbal stem cells. A 13 kbp mouse ALDH3 promoter fragment containing >12 kbp of 5' flanking sequence as well as exon 1 and 29 bp of intron 1 directed CAT reporter gene expression to the stomach, bladder and cornea. By contrast, when driven by an approximate 4 kbp promoter fragment containing 1050 bp of 5' flanking sequence, exon 1, intron 1 (3 kbp) and 7 bp of exon 2 expression of the CAT reporter gene was confined to the corneal epithelial cells, except for very low levels in the liver. Thus tissue-specific expression of the ALDH3 gene is determined by positive and negative elements in the 5' flanking region and intron 1. This is the first identification of a corneal-specific promoter and opens the door to genetic engineering in the cornea. The TKT and ALDH3 genes have numerous inducible cis-control consensus sequences in their 5'flanking regions consistent with the possibility that induction by the environment (i.e. uv light) contributes significantly to their high expression in the cornea. This fits with our observation that ALDH3 and TKT expression is minimal in the mouse corneal epithelial cells until after eye opening 14 days after birth. Moreover, corneal TKT was induced several fold by exposing mice raised in the dark for 25 days after birth to light and in explanted eyes exposed to light derived from new-born mice. The use of inductive mechanisms involving the environment differs from the strictly developmental control processes used by the lens for the high, preferential expression of crystallin genes. We have also studied Pax-6 expression in the mouse cornea. In general, Pax-6 is preferentially expressed in the limbal stem cells and dividing anterior epithelial cells. This is inversely related to the expression pattern of ALDH3, raising the possibility that Pax-6 represses ALDH3 gene expression. The heterozygote small eye Dickey mouse, which has a deletion including the Pax-6 gene, has a monolayered corneal epithelium, supporting the idea that Pax-6 is required for the proliferation and stratification of the epithelial cells during development. A similar interpretation applied to the healing, normal corneal epithelial cells. Finally, initial experiments have shown that an approximate 70 kDa protein comprises at least 50% if not more of the water-soluble protein of the cornea of the zebrafish. Amino acid sequences obtained from 4 tryptic peptides derived from the 70 kDa protein purified by polyacrylamide gel electrophoresis indicated that it is either identidal or related to gelsolin, an actin binding protein. A gelsolin mutation in humans has been associated with lattice type II corneal dystrophy. Experiments are in progress to determine the role of gelsolin in the zebrafish cornea.